Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR ...

Clinical Cancer Research

Human Cancer Biology

Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 Are Localized Primarily to the Vasculature in Human Primary Solid Cancers Neil R. Smith1, Dawn Baker1, Neil H. James1, Kirsty Ratcliffe1, Martin Jenkins2, Susan E. Ashton1, Graham Sproat1, Ruth Swann1, Neil Gray1, Anderson Ryan1, Juliane M. Jürgensmeier1, and Chris Womack2

Abstract Purpose: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. Experimental Design: VEGFR-2– and VEGFR-3–specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. Results: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. Conclusions: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3–targeted therapy. Clin Cancer Res; 16(14); 3548–61. ©2010 AACR.

Vascular endothelial growth factor (VEGF) signaling is key to physiologic and pathologic angiogenesis and lymphangiogenesis, and is an established target in the development of anticancer therapeutics. VEGF-A is an important driver of the neovascular growth required to support solid tumor progression (1). The primary signaling receptor for VEGF-A is VEGF receptor (VEGFR)-2 (Flk-1, KDR; refs. 2, 3), and activation of VEGFR-2 by VEGF-A on endothelial tip and stalk cells directs the migration and extension of sprouting vessels, respectively (4). VEGFR-3 (Flt-4) is the

Authors' Affiliations: 1 Cancer Bioscience and 2 Oncology Clinical Development, AstraZeneca, Cheshire, United Kingdom Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Neil R. Smith, Cancer Bioscience, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom. Phone: 44-1625-233731; Fax: 44-1625-510097; E-mail: Neil. [email protected]. doi: 10.1158/1078-0432.CCR-09-2797 ©2010 American Association for Cancer Research.

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receptor for VEGF-C and VEGF-D (5). Expression of VEGFR-3 is largely confined to lymphatic endothelium in adult tissues (6), and its activation induces lymphangiogenesis (7). VEGFR-3 was recently also found expressed on the endothelial tip cells of angiogenic tumor vasculature, and blockade of the receptor in tumor models induces blood vessel endothelial cell sprouting and vessel branching (8). Agents that inhibit VEGF signaling and are already registered for the treatment of cancer include the VEGF-A neutralizing antibody bevacizumab (9) and small-molecule multikinase inhibitors sorafenib, sunitinib, and pazopanib (10, 11). Other small-molecule VEGFR inhibitors under evaluation in clinical trials include vandetanib and cediranib. Cediranib is a potent inhibitor of VEGF signaling, targeting VEGFR-1, VEGFR-2, and VEGFR-3 and angiogenesis (12). Recent publications have shown that VEGFR-3 inhibition by cediranib reduces lymphangiogenesis as well as the incidence of lymphatic metastases in vivo (13, 14). The combined blockade of VEGFR-2 and VEGFR3 has an additive antiangiogenic and antitumor effect

VEGFR-2 and VEGFR-3 Status of Human Solid Tumors

Translational Relevance Vascular endothelial growth factor (VEGF) signaling inhibitors have shown clinical efficacy in a range of solid tumor types, including colorectal, lung, and breast cancer. A better understanding of the expression and localization of VEGF receptors is needed to understand the potential for direct antitumor effects from VEGF signaling inhibitors in the clinic and to help identify patients who might gain greatest benefit from treatment. Our findings indicate that VEGFR-2 and VEGFR-3 are primarily localized to tumor vessels in a broad range of solid tumors, suggesting that tumor endothelial cells and not tumor cells are the likely primary target for anti–VEGFR-2 and anti–VEGFR-3 therapy in cancer. Furthermore, the expression of VEGFR-3, as well as VEGFR-2, on tumor blood vessels suggests that dual VEGFR-2/VEGFR-3 inhibitors may have additional antiangiogenic benefits in some patients.

in vivo over inhibition of VEGFR-2 alone (8). In addition to reducing lymphangiogenic metastases, dual VEGFR-2 and VEGFR-3 inhibitors may improve the antiangiogenic response in patients. An understanding of the VEGFR-2 and VEGFR-3 status of human tumors may help to identify patients who would benefit most from treatment with dual VEGFR-2 and VEGFR-3 inhibitors and clarify the clinical mechanism of action of these agents. Presently, there is no clear agreement from the literature on the location (vasculature and/or cancer cells) or extent of expression of either VEGFR-2 or VEGFR-3 in human solid tumors. For example, using immunohistochemistry, VEGFR-2 has been detected on tumor vessels supporting human colorectal (CRC; ref. 15), breast (BC; refs. 16, 17), and non–small cell lung carcinomas (NSCLC; refs. 18, 19). However, tumor cell expression has also been reported in 40% to 100% of CRC (20, 21), BC (22–24), and NSCLC (18, 19, 25). Similarly, some reports confine VEGFR-3 to tumor vessels, primarily lymphatic but also blood endothelium in CRC (26), BC (27–30), and NSCLC (19, 31–33), whereas others localize the receptor to tumor cells [CRC (34), BC (23, 35), and NSCLC (19, 25, 32, 33, 36, 37)]. Recently, Petrova et al. (38) have provided compelling evidence that poor antibody specificity is likely to be responsible for some of the claims that VEGFR-3 is found on human tumor cells (38). Using a validated antibody, they confirm that VEGFR-3 is confined primarily to blood and lymphatic vessels in solid tumors (38). This report aimed to resolve the issues around the relative extent and location of VEGFR-2 and VEGFR-3 expression in human primary solid tumors and, in doing so, better define the clinical mechanism of action of VEGFtargeted therapy. To do this, we determined the crossreactivity of human VEGFR antibodies using multiple

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screening platforms and identified two antibodies, 55B11 (VEGFR-2; Cell Signaling Technology) and AF349 (VEGFR-3; R&D Systems), with credible specificity. These were used in immunohistochemical and coimmunofluorescent analyses of archival human solid tumor samples to understand the expression of the two receptors between (a) tumor endothelium and malignant cells and (b) tumor and normal endothelium and investigate the relative localization of VEGFR-2 and VEGFR-3 on tumor vasculature.

Materials and Methods Antibodies The following antibodies were used: custom rabbit polyclonal antibody raised to COOH-terminal peptide of mouse CD31 (CHG-CD31-P1; AstraZeneca); mouse monoclonal antibodies to human CD31 (JC70A; Dako), α-smooth muscle actin (α-SMA; 1A4; Sigma), phosphotyrosine (P-Tyr-100; Cell Signaling Technology), and rabbit glyceraldehyde-3-phosphate dehydrogenase (AM4300; Ambion); rabbit monoclonal against human VEGFR-2 (55B11); goat polyclonal against human VEGFR-3 (AF349); and rabbit polyclonal against human LYVE-1 (102-PA50AG; Reliatech). Other VEGFR antibodies are listed in Supplementary Table S1. Cell lines Cell lines were maintained at 37°C with 5% CO2: HT-29 (obtained from the European Collection of Animal Cell Cultures) in DMEM with 5% fetal bovine serum (FBS) and 1% nonessential amino acids; Colo-205 (European Collection of Animal Cell Cultures) in RPMI 1640/10% FBS; A549 (American Type Culture Collection) in DMEM/10% FBS/10% M1; PC-9 (a kind gift from Dr. K Nishio, National Cancer Centre of Japan) in RPMI 1640/10% FBS/10% M1; MCF-7 (Imperial Cancer Research Fund) in DMEM/10% FCS; aortic VSMC (C-12533; Promocell) in Promocell VSMC growth media (C-22062); M-07e (Deutsche Sammlung von Mikroorganismen und Zellkulturen) in RPMI 1640/10% FBS/10% M1/interleukin-3 (10 ng/mL)/granulocyte macrophage colony-stimulating factor (10 ng/mL); PAE (Ludwig Institute for Cancer Research) in Ham's F12/ 10% FBS/L-glutamine; and NIH-3T3 (Jefferson Cancer Institute) in DMEM/10% FBS/L-glutamine. Full-length human cDNA for VEGFR-1, VEGFR-2, and CSF1R was PCR amplified from nm_002019.3, nm_002253.2, and nm_005211.3, respectively, using the following primer pairs: VEGFR-1, 5′-GTTTAACTTTAAGAAGGAGATATAACCATGGTCAGCTACTGGGACACCGGGG-3′ (forward) and 5′-CTATAGGTCCTCCTCTGATATTAGCTTTTGCTCGATGGGTGGGGTGGAGTACAGGACC-3′ (reverse); VEGFR-2, 5′-ATTTAACTTTAAGAAGGAGATATAACCCAGAGCAAGGTGCTGCTGGCCGTCGCCC-3′ (forward) and 5′-TCTATAGGTCCTCCTCTGATATTAGCTTTTGCTCAACAGGAGGAGAGCTCAGTGTGG-3′ (reverse); and CSF1R, 5′-TCATAGGTCCTCCTCTGATATTAGCTTTTGCTCGCAGAACTGATAGTTGTTGGGCTGC-3′


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(forward) and 5′-ATTTAACTTTAAGAAGGAGATATAACCATGGGCCCAGGAGTTCTGCTGCTCCTGC-3′ (reverse). VEGFR-3 (cDNA cloned into pDONR221), accession number nm_182925, was obtained from GeneArt. DNA fragments were subcloned into the retroviral vector pBIN CIP SIN via Gateway technology (Invitrogen) and cotransfected with constructs expressing the viral envelope protein VSVG (Clontech) into a packaging cell line, Phoenix-Hek 293 (Garry P. Nolan, Stanford University School of Medicine). Retroviral particles were used to infect NIH-3T3 and PAE cells, and stable lines were selected for resistance to puromycin. Tumor xenografts Tumor xenograft tissue was derived from experiments conducted with licenses issued under the UK Animals (Scientific Procedures) Act 1986 and after local ethical review and approval. Cell lines were maintained in the recommended growth medium and implanted s.c. into the left flank of immunocompromised mice: nude, scid, or scidbg (see Supplementary Table S2 for details). Tumors were grown to ∼1 cm3 volume and then collected and fixed in formalin for 24 hours before being embedded in paraffin. Patient tissue samples Formalin-fixed, paraffin-embedded (FFPE) human primary cancer resection samples and tissue microarrays (TMA) from patients with untreated human primary bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid cancers and matched adjacent normal tissue (for colorectal, lung, and breast cancers) were sourced under approved legal contract from three commercial tissue suppliers (Asterand, Cytomyx, and TriStar Technology Group) and a hospital tissue bank (Wales Cancer Bank). Appropriate consents, licensing, and ethical approval were obtained for this research. The suitability of each specimen for immunohistochemical analyses was determined by pathology assessment of tissue morphology and preservation (H&E) and the general extent of antigen preservation (CD31 and p-Tyr immunostains). Western blot analysis Subconfluent (70-80%) cell lines were scraped into icecold lysis buffer [20 mmol/L Tris (pH 7.5), 137 mmol/L NaCl, 10% glycerol, 1% NP40, 0.1% SDS, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 protease inhibitor tablet/25-mL buffer (Boehringer Ingelheim)], incubated on ice for 30 minutes, aspirated, and cleared by centrifugation. Frozen tumor xenograft tissues were homogenized in ice-cold lysis buffer using an Ultraturrax T25 homogenizer (Janke and Kunkel) before incubation on ice. Lysate samples (250 μg) were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane (Invitrogen) by Western blotting. Membranes were blocked in TBS containing 0.05% Tween (TBST) and 5% nonfat dry milk (Marvel) for 1 hour at room temperature and then incubated for 16 hours at 4°C in VEGFR-2

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antibodies (55B11, A3, sc-504, and sc-315; diluted 1:1,000 in block), VEGFR-3 antibody (AF349; 1:1,000), CD31 antibody (CHG-CD31-P1; 1:500), or glyceraldehyde-3-phosphate dehydrogenase antibody (AM4300; 1:10,000). Immunoblots were incubated in a 1:2,000 dilution of horseradish peroxidase–conjugated anti-rabbit (New England Biolabs), anti-mouse (New England Biolabs), or anti-goat (Dako) antibody and visualized using the SuperSignal West Pico Substrate method of detection (Perbio Science). Immunohistochemistry and immunocytochemistry All incubations were at room temperature and washes were done with TBST. FFPE tissues sectioned at 4 μm onto slides were dewaxed and rehydrated. Antigen retrieval was done in a RHS-1 microwave vacuum processor (Milestone) at 110°C for 5 minutes; in pH 6 retrieval buffer (S1699; Dako) for p-Tyr, CD31, and VEGFR-3; or in pH 9 retrieval buffer (S2367; Dako) for VEGFR-2. Endogenous biotin was blocked using an avidin-biotin kit (SP-2002; Vector Laboratories, Inc.), endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes, and nonspecific binding sites were blocked with serumfree protein block (X0909; Dako) for 20 minutes. Primary antibodies to p-Tyr (P-Tyr-100), CD31 (JC70A and CHGCD31-P1), VEGFR-2 (55B11), and VEGFR-3 (AF349) were diluted 1:500, 1:500, 1:600, and 1:200, respectively, in antibody diluent (S0809; Dako) and incubated with sections for 1 hour. Either mouse Envision secondary (K4007; Dako) for p-Tyr and CD31, rabbit Envision secondary (K4003; Dako) for VEGFR-2 and CD31 (CHG-CD31P1), or biotinylated rabbit anti-goat immunoglobulin antibodies (E0466, diluted 1:400 in TBST; Dako), followed by Vectastain Elite ABC solution (PK-6100; Vector Laboratories), for VEGFR-3 were added for 30 minutes each. Sections were developed in diaminobenzidine for 10 minutes (K3466; Dako) and counterstained with Carazzi's hematoxylin. Appropriate no primary antibody and isotype controls were done for each antibody. For immunocytochemical analyses, subconfluent cells were transferred to 10% neutral buffered formalin for 24 hours at 4°C. Cells were centrifuged to a pellet, washed in 80% ethanol, processed into 1% agarose, and embedded into paraffin blocks. Cell pellets were sectioned at 4 μm, and 55B11 and AF349 antibodies were evaluated using the immunohistochemical conditions without the antigen retrieval. Immunofluorescence Immunofluorescence was done on dewaxed and rehydrated FFPE sections. Antigen retrieval was carried out as above in pH 9 retrieval buffer. After blocking in 20% horse serum, sections were incubated for 1 hour with antibody pairs diluted in serum: 55B11 (1:20, VEGFR-2) and AF349 (1:20, VEGFR-3); 55B11 and JC70A (1:20, CD31); AF349 (1:20) and JC70A (1:20); 55B11 (1:20) and 1A4 (1:1,000, α-SMA); AF349 (1:20) and 1A4 (1:1,000); and JC70A (1:20) and 102-PA50AG (1:60). Appropriate secondary


VEGFR-2 and VEGFR-3 Status of Human Solid Tumors

antibody pairs combined 1:800 in serum were added for 30 minutes: 55B11 was detected with donkey antirabbit IgG conjugated to Alexa Fluor 555 or Alexa Fluor 488 (A31572 or A21206; Molecular Probes); AF349 with donkey anti-goat IgG Alexa Fluor 555 (A21432; Molecular Probes); NP056 with donkey anti-mouse IgG Alexa Fluor 488 or Alexa Fluor 555 (A21202 or A31570; Molecular Probes); 1A4 with donkey anti-mouse IgG Alexa Fluor 488; and 102-PA50AG with donkey anti-rabbit IgG Alexa Fluor 488. Sections were counterstained with ProLong Gold antifade reagent with 4′,6-diamidino-2phenylindole (P36931; Molecular Probes), and fluorescent images were scanned and captured using a MIRAX scan (Carl Zeiss). Image analysis and pathology review The mean number of VEGFR-2– and CD31-positive vessels per mm2 viable tumor for each xenograft tumor model was quantified as described previously (39). The percentage of VEGFR-2–positive vessels per tumor model was calculated as follows: number VEGFR-2 vessels per mm2/number of CD31 vessels per mm2 × 100. All human tissue sections were scored by a pathologist using a light microscope. A subjective reporting procedure was implemented, recording location (tumor cell, normal epithelium and vessel) and intensity [− (none), + (weak), ++ (medium), and +++ (high)] of VEGFR-2, VEGFR-3, and CD31 staining. Statistical analysis For each tissue sample, the maximum VEGFR-2 or VEGFR-3 staining intensity was recorded on an ordinal scale (0, +, ++, +++). Separate intensities were recorded for the epithelium and endothelium in each sample where possible. A Wilcoxon signed-rank test was used to compare the relative intensity of staining between (a) tumor epithelium and endothelium in a sample and (b) tumor and normal endothelium for matched pairs of tumor and normal tissue. The Bonferroni-Holm procedure was used to take account of the multiple tests being carried out in several tissue types and to control the family-wise error rate at 0.05 for each of VEGFR-2 and VEGFR-3. Those tests where the null hypothesis (of no difference between groups) would be rejected are highlighted in Table 1. The proportion of samples with positive VEGFR staining (+, ++, or +++) was also estimated in each tissue type, and an exact 95% binomial confidence interval was given.

Results Evaluation of VEGFR antibodies A total of 12 antibodies, raised to either human VEGFR-2 (n = 8) or VEGFR-3 (n = 4), were selected for evaluation based on the supplier's recommendation and/or a literature precedence for their use in immunohistochemistry (Supplementary Table S1). Of the eight VEGFR-2 antibodies tested, only 55B11 was VEGFR-2 specific and qualified for use in immunohisto-

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chemistry (Supplementary Table S1). By Western blot analysis, 55B11 detected a 230-kDa doublet band indicative of fully and partially glycosylated VEGFR-2 (40) in NIH-3T3 and PAE cells engineered to overexpress human VEGFR-2 but detected nothing in the untransfected parental cell lines (Fig. 1A). A band ∼150 kDa was also detected in VEGFR-2–overexpressing PAEs, which is likely to be unglycosylated VEGFR-2 protein (40). These data were confirmed by immunocytochemical analysis of FFPE parental and VEGFR-overexpressing PAE cells (Fig. 1B). 55B11 did not cross-react with VEGFR-2–related human kinases [VEGFR-1, VEGFR-3, platelet-derived growth factor receptor-β (PDGFR-β), c-Kit, or CSF1R] as determined by Western blot analysis of PAE and NIH-3T3 cells engineered to overexpress human VEGFR-1 (150 kDa), VEGFR-3 (75/120/195 kDa), or CSF1R (130/150 kDa) and human aortic VSMCs and M-07e, which express high levels of endogenous PDGFR-β (180 kDa; ref. 41) and cKit (145 kDa; ref. 42), respectively (Fig. 1A). Furthermore, 55B11 cleanly detected a 230-kDa VEGFR-2 doublet band by Western blotting in more complex heterogeneous protein lysates (e.g., human cancer xenograft lysates; Fig. 2C). In our hands, the VEGFR-2 antibodies 55B11, A3, sc-315, and sc-504 commonly used to investigate the expression of VEGFR-2 in human tumors (18–24), did not qualify as specific. All three detected multiple bands, running at different sizes to that expected for VEGFR-2, in lysates prepared from cell lines expressing VEGFR family members (Fig. 1A) and tumor xenografts and matched parental tumor cell lines (Fig. 2C). Because cross-reactivity of these antibodies with other proteins could potentially confound any analysis of VEGFR-2 expression by immunohistochemistry, they were not used in the analysis of human tumor samples. Of four antibodies evaluated, both AF349 and 9D9F9 were specific for VEGFR-3 and suitable for immunohistochemistry (Supplementary Table S1). AF349 was selected for further analyses over 9D9F9, as this was raised in goat and was therefore more suitable for use in coimmunofluorescence experiments with mouse monoclonal antibodies to CD31 and α-SMA. AF349 detected a 195-kDa protein, representing immature (nonproteolytically cleaved) VEGFR-3, in VEGFR-3–overexpressing PAE and NIH-3T3 cells, which was absent from the parental cell lines (Fig. 1A). Bands running at approximately 120 and 75 kDa were also detected in VEGFR-3–overexpressing PAEs. These represent the denatured mature protein, as fully processed mature VEGFR-3 consists of 120- and 75-kDa proteins linked by a disulfide bridge (43). In addition, AF349 did not cross-react with VEGFR-3–related kinases VEGFR-1, VEGFR-2, PDGFR-β, c-Kit, or CSF1R (Fig. 1A). The specificity of AF349 for VEGFR-3 was confirmed by immunocytochemical analysis of engineered PAE cells (Fig. 1B). VEGFR-2 expression in human tumor xenografts grown in immunocompromised mice TMAs were generated from 29 histologically diverse human tumor xenografts grown to ∼1 cm3. Serial sections


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Table 1. Maximum VEGFR-2 and VEGFR-3 histopathology intensity scores for human tumors Tissue

VEGFR-2 n

T/N Endo 0


Bladder cancer* BC* CRC* Head and neck cancer* Liver cancer* NSCLC* Melanoma* Ovarian cancer* Pancreatic cancer* Prostate cancer* Renal cancer* Stomach cancer* Thyroid cancer* BC‡ Matched normal breast‡ CRC‡ Matched normal colorectal‡ NSCLC‡ Matched normal lung‡

+

++ +++ 0

VEGFR-3

T Endo T Endo n vs. T cell vs. N (P) Endo (P) +/++/ Prop Exact 95% CI +++ +ve

T cells/ N Epi

Endo positives

T/N Endo

T cells/ N Epi

0

+

++ +++ 0

Endo positives

T Endo T Endo vs. T cell vs. N (P) Endo (P) +/++/ Prop Exact 95% CI +++ +ve

10 36 41 11

0 2 0 0

0 2 2 1

4 18 15 4

6 14 24 6

10 36 41 11

0 0 0 0

1.000 0.944 1.000 1.000

(0.692-1.000) (0.813-0.993) (0.914-1.000) (0.715-1.000)

0.0020†